Regenerative granulopoietic failure is common in patients infected by HIV-1, a phenomenon we and others have attributed to dysfunction of HIV-1-infected bone marrow accessory cells; microvascular endothelial cells and mononuclear phagocytes in particular.(1) We have found that HIV-1 infected bone marrow stromal cells (infected ex-vivo or in-vivo) are less supportive of granulocyte-macrophage progenitor cell (CFU-GM) survival in co-cultures and, after stimulation with IL-1 produce significantly lower amounts of IL-6, GM-CSF and G-CSF than do uninfected stromal cells. We have also found that while infected stromal cells have a reduced capacity to support granulopoiesis, they are more supportive of growth/survival of lymphoma cells with the germinal center phenotype. Using vesicular stomatitis virus-G (VSV-G) pseudotyped HIV-1 vectors with inactivating mutations of various HIV-1 regulatory/accessory genes to transduce normal bone marrow stromal cells, we have determined that the two suppressive effects of HIV-1 on granulopoietic support function require HIV-1 vpr. The aims of this proposal are designed to test the hypothesis that HIV-1 vpr is necessary and sufficient to account for the observed changes in hematopoietic support function of bone marrow stroma. Our first aim is to identify the molecular points of suppression of GM-CSF, G-CSF, and IL-6 gene expression by HIV-1 and HIV-1 vpr and to determine whether induction of these factors by TNF-alpha and CD40 ligand (CD40L) is similarly suppressed. Our second aim is to test the cause-and-effect relationship between GM-CSF, IL-6 and G-CSF inhibition and the inhibitory effects of HIV-1-infected bone marrow stromal cells on survival of CFU-GM. Our third aim is to use both retroviral mediated gene transfer of and tet-responsive vectors expressing HIV-1 vpr and other accessory proteins to determine the sufficiency of HIV-1 vpr in; (a) blunting bone marrow stromal cell responses to IL-1 (and other inducing factors including CD40L and TNF-alpha), (b) reducing the capacity of stroma to support CFU-GM, and (c) enhancing the survival of lymphoma cells with the germinal center phenotype. Results of these studies should clarify the molecular pathogenesis of regenerative granulopoietic failure in HIV-1 seropositive patients and may also reveal determinants of lymphoma progression in this clinical context.